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- PDB-5ue5: proMMP-7 with heparin octasaccharide bound to the catalytic domain -

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Basic information

Entry
Database: PDB / ID: 5ue5
TitleproMMP-7 with heparin octasaccharide bound to the catalytic domain
ComponentsMatrilysinMMP7
KeywordsHYDROLASE / glycan complex with protein / enzyme complex with heparin oligosaccharide / zymogen / allosteric effector site
Function / homology
Function and homology information


matrilysin / membrane protein intracellular domain proteolysis / Assembly of collagen fibrils and other multimeric structures / Activation of Matrix Metalloproteinases / membrane protein ectodomain proteolysis / Collagen degradation / collagen catabolic process / extracellular matrix disassembly / extracellular matrix / Degradation of the extracellular matrix ...matrilysin / membrane protein intracellular domain proteolysis / Assembly of collagen fibrils and other multimeric structures / Activation of Matrix Metalloproteinases / membrane protein ectodomain proteolysis / Collagen degradation / collagen catabolic process / extracellular matrix disassembly / extracellular matrix / Degradation of the extracellular matrix / extracellular matrix organization / metalloendopeptidase activity / metallopeptidase activity / endopeptidase activity / Extra-nuclear estrogen signaling / positive regulation of cell migration / serine-type endopeptidase activity / proteolysis / extracellular space / extracellular exosome / zinc ion binding / extracellular region
Similarity search - Function
Peptidoglycan binding-like / Peptidase M10A, cysteine switch, zinc binding site / Matrixins cysteine switch. / Putative peptidoglycan binding domain / Peptidase M10A / Peptidase M10A, catalytic domain / Peptidase M10, metallopeptidase / Matrixin / PGBD-like superfamily / Peptidase, metallopeptidase ...Peptidoglycan binding-like / Peptidase M10A, cysteine switch, zinc binding site / Matrixins cysteine switch. / Putative peptidoglycan binding domain / Peptidase M10A / Peptidase M10A, catalytic domain / Peptidase M10, metallopeptidase / Matrixin / PGBD-like superfamily / Peptidase, metallopeptidase / Zinc-dependent metalloprotease / Collagenase (Catalytic Domain) / Collagenase (Catalytic Domain) / Metallopeptidase, catalytic domain superfamily / Neutral zinc metallopeptidases, zinc-binding region signature. / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
MethodSOLUTION NMR / torsion angle dynamics
AuthorsFulcher, Y.G. / Prior, S.H. / Linhardt, R.J. / Van Doren, S.R.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM057289 United States
Citation
Journal: Structure / Year: 2017
Title: Glycan Activation of a Sheddase: Electrostatic Recognition between Heparin and proMMP-7.
Authors: Fulcher, Y.G. / Prior, S.H. / Masuko, S. / Li, L. / Pu, D. / Zhang, F. / Linhardt, R.J. / Van Doren, S.R.
#1: Journal: Structure / Year: 2015
Title: Charge-Triggered Membrane Insertion of Matrix Metalloproteinase-7, Supporter of Innate Immunity and Tumors.
Authors: Prior, S.H. / Fulcher, Y.G. / Koppisetti, R.K. / Jurkevich, A. / Van Doren, S.R.
History
DepositionDec 29, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 19, 2017Provider: repository / Type: Initial release
Revision 1.1Sep 27, 2017Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.2Jan 1, 2020Group: Author supporting evidence / Data collection
Category: pdbx_audit_support / pdbx_nmr_software / pdbx_nmr_spectrometer
Item: _pdbx_audit_support.funding_organization / _pdbx_nmr_software.name / _pdbx_nmr_spectrometer.model
Revision 2.0Jul 29, 2020Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Derived calculations / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / database_PDB_caveat / entity / pdbx_branch_scheme / pdbx_chem_comp_identifier / pdbx_entity_branch / pdbx_entity_branch_descriptor / pdbx_entity_branch_link / pdbx_entity_branch_list / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_struct_assembly_gen / pdbx_struct_conn_angle / pdbx_unobs_or_zero_occ_atoms / pdbx_validate_chiral / struct_asym / struct_conn / struct_conn_type / struct_site / struct_site_gen
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.auth_seq_id / _atom_site.label_asym_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.label_entity_id / _atom_site.type_symbol / _chem_comp.mon_nstd_flag / _chem_comp.name / _chem_comp.type / _pdbx_struct_assembly_gen.asym_id_list / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_unobs_or_zero_occ_atoms.auth_asym_id / _pdbx_unobs_or_zero_occ_atoms.auth_seq_id / _pdbx_unobs_or_zero_occ_atoms.label_asym_id / _pdbx_validate_chiral.auth_asym_id / _pdbx_validate_chiral.auth_seq_id / _struct_conn.conn_type_id / _struct_conn.id / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn_type.id
Description: Carbohydrate remediation / Provider: repository / Type: Remediation

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Matrilysin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)30,1286
Polymers27,5891
Non-polymers2,5395
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)16 / 200structures with the least restraint violations
RepresentativeModel #1target function

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Components

#1: Protein Matrilysin / MMP7 / Matrin / Matrix metalloproteinase-7 / MMP-7 / Pump-1 protease / Uterine metalloproteinase


Mass: 27589.139 Da / Num. of mol.: 1 / Mutation: E195A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MMP7, MPSL1, PUMP1 / Plasmid: pET28A / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P09237, matrilysin
#2: Polysaccharide 2-O-sulfo-alpha-L-idopyranuronic acid-(1-4)-2-deoxy-6-O-sulfo-2-(sulfoamino)-alpha-D-glucopyranose- ...2-O-sulfo-alpha-L-idopyranuronic acid-(1-4)-2-deoxy-6-O-sulfo-2-(sulfoamino)-alpha-D-glucopyranose-(1-4)-2-O-sulfo-alpha-L-idopyranuronic acid-(1-4)-2-deoxy-6-O-sulfo-2-(sulfoamino)-alpha-D-glucopyranose-(1-4)-2-O-sulfo-alpha-L-idopyranuronic acid-(1-4)-2-deoxy-6-O-sulfo-2-(sulfoamino)-alpha-D-glucopyranose-(1-4)-2-O-sulfo-alpha-L-idopyranuronic acid-(1-4)-2-deoxy-6-O-sulfo-2-(sulfoamino)-alpha-D-glucopyranose


Type: oligosaccharide / Mass: 2327.897 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
WURCS=2.0/2,8,7/[a2122h-1a_1-5_2*NSO/3=O/3=O_6*OSO/3=O/3=O][a2121A-1a_1-5_2*OSO/3=O/3=O]/1-2-1-2-1-2-1-2/a4-b1_b4-c1_c4-d1_d4-e1_e4-f1_f4-g1_g4-h1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNSO36SO3]{[(4+1)][a-L-IdopA2SO3]{[(4+1)][a-D-GlcpNSO36SO3]{[(4+1)][a-L-IdopA2SO3]{[(4+1)][a-D-GlcpNSO36SO3]{[(4+1)][a-L-IdopA2SO3]{[(4+1)][a-D-GlcpNSO36SO3]{[(4+1)][a-L-IdopA2SO3]{}}}}}}}}LINUCSPDB-CARE
#3: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca
#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
111isotropic13D 1H-15N NOESY
121isotropic1TROSY-detected titration
232isotropic1PREs by PROJECT-CPMG TROSY
344isotropic1solvent PREs, 1H relaxation detected via TROSY
455isotropic13D HN(CA)CB
465isotropic13D CBCA(CO)NH

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Sample preparation

Details
TypeSolution-IDContentsDetailsLabelSolvent system
solution1300 uM [U-99% 15N] proMMP-7, 450 uM heparin dp8, 93% H2O/7% D2OproMMP-7 + heparin dp8proMMP-7 + heparin dp893% H2O/7% D2O
solution2300 uM [U-99% 2H/15N] proMMP-7, 450 uM heparin dp8-TEMPO, 93% H2O/7% D2Oreducing end of heparin dp8 was substituted with TEMPO spin labelproMMP-7 + spin-labeled heparin dp893% H2O/7% D2O
solution3300 uM [U-99% 2H/15N] proMMP-7, 450 uM heparin dp8, 93% H2O/7% D2Odeuterated proMMP-7 + heparin dp8deuterated proMMP-7 + heparin dp893% H2O/7% D2O
solution4300 uM [U-99% 2H/15N] proMMP-7, 450 uM heparin dp4, 93% H2O/7% D2OproMMP-7 +/- heparin dp4 The free state of proMMP-7 and its complex with heparin dp4 were each measured without and with the addition of 0.3 mM Gd-EDTAproMMP-7 +/- heparin dp493% H2O/7% D2O
solution5300 uM [U-100% 13C; U-100% 15N; U-80% 2H] proMMP-7, 93% H2O/7% D2OCollected and interpreted for BMRB ID 25485 and Prior et al. (2015; 10.1016/j.str.2015.08.013proMMP-7, free state93% H2O/7% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
300 uMproMMP-7[U-99% 15N]1
450 uMheparin dp8natural abundance1
300 uMproMMP-7[U-99% 2H/15N]2
450 uMheparin dp8-TEMPOnatural abundance2
300 uMproMMP-7[U-99% 2H/15N]3
450 uMheparin dp8natural abundance3
300 uMproMMP-7[U-99% 2H/15N]4
450 uMheparin dp4natural abundance4
300 uMproMMP-7[U-100% 13C; U-100% 15N; U-80% 2H]5
Sample conditions
Conditions-IDDetailsIonic strengthLabelpHPressure (kPa)Temperature (K)
120 mM imidazole, 10 mM CaCl2, 150 mM NaCl, 20 uM ZnCl2, 10 mM 2-mercaptoethanol180 mMproMMP-7 + heparin dp86.6 1 atm310 K
220 mM imidazole, 10 mM CaCl2, 150 mM NaCl, 20 uM ZnCl2, 10 mM 2-mercaptoethanol Measurements without and with addition of 10 mM ascorbate180 mMproMMP-7 + spin-labeled heparin dp86.6 1 atm310 K
3+/- 450 uM hep dp4 and +/- 300 uM GdEDTA 20 mM imidazole, 10 mM CaCl2, 150 mM NaCl, 20 uM ZnCl2, 10 mM 2-mercaptoethanol180 mMproMMP-7 +/- hep dp4 +/- GdEDTA6.6 1 atm310 K
4Conditions used for assigning the peaks and determining the solution structure (PDB ID: 2MZE) 20 mM imidazole, 10 mM CaCl2, 20 uM ZnCl2, 10 mM 2-mercaptoethanol30 mMproMMP-7, free state6.6 1 atm310 K

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NMR measurement

NMR spectrometerType: Bruker AVANCE III / Manufacturer: Bruker / Model: AVANCE III / Field strength: 800 MHz / Details: TCI cryoprobe

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Processing

SoftwareName: SYBYL / Version: X 2.1.1 / Classification: refinement
NMR software
NameVersionDeveloperClassification
CYANA2.1Guntert, Mumenthaler and Wuthrichstructure calculation
SparkyGoddarddata analysis
AnalysisCCPNchemical shift assignment
TopSpinBruker Biospinprocessing
SYBYLX 2.1.1Tripos / Certararefinement
RefinementMethod: torsion angle dynamics / Software ordinal: 1
Details: 15000 steps of refinement. Explicit distance restraints to the TEMPO spin-labeled reducing end of heparin dp8 were obtained from the PREs (T2) using an equation from Battiste and Wagner ...Details: 15000 steps of refinement. Explicit distance restraints to the TEMPO spin-labeled reducing end of heparin dp8 were obtained from the PREs (T2) using an equation from Battiste and Wagner (2000, Biochemistry) simplified by WHTC greater than1 to the form r equal to 4KTC/T2 given by Koppisetti et al. (2014, Nature Comms.). The rotational correlation time TC of the protein-heparin complexes was obtained from amide 15N cross-correlation rates nxy (Liu and Prestegard, 2008, J. Magn. Reson.) interpreted by the spectral density expression used in the TRACT approach (Lee et al., 2006. J. Magn. Reson.). Upper bounds were set at 15% above the distance estimate. An intermolecular NOE from an arginine backbone amide from 2H/15N-labeled enzyme to the anomeric proton H1 of GlcNS6S 5 or 7 had an upper bound of 6 A. Lower bounds were implicitly at van der Waals distance. Ambiguous distance restraints from any residue of heparin dp8 to explicit protein amide groups were applied on the basis of amide chemical shift perturbations from heparin dp8 or protection of amides by heparin dp4 from line broadening by Gd.EDTA with deltaT2 greater than 37/sec. Ambiguous distance restraints from any residue of heparin dp8 to lysine amino or arginine ureido groups were also applied on the basis of mutations that impaired activation of proMMP-7 by heparin dp16. The combination of the intermolecular distance restraints from the explicit PRE and NOE measurements and the ambiguous sources were used to dock coordinates of heparin dp8 with the NMR solution structure of human proMMP-7 of Prior et al. (2015, Structure). To enable molecular flexibility widely through the proMMP-7 and heparin dp8 chains while maintaining the structural integrity of the proMMP-7 during the restrained docking simulations, CYANA 2.1 (Guntert and Buchner, 2015, J. Biomol. NMR) was utilized in concert with the intramolecular proMMP-7 NOE-derived distance restraints and chemical shift-derived dihedral restraints of the NMR structure (Prior et al., 2015, Structure). CYANA topology files describing the sugar monomers were derived from topology files curated by the Automated Topology Builder (Malde et al., 2011, J. Chem. Theory Comput.) and based on accession number 9804 for 2-O-sulfo-alpha-L-idopyranuronic acid (IDS or IdoA2S) and 9778 for N,O6-disulfo-glucosamine (SGN or GlcNS6S). The globally flexible docking simulations required the proMMP-7 polypeptide to be linked to the calcium and zinc ions and heparin dp8 chains via tethers of non-interacting pseudoatoms. With this tethering and flexible structural integrity enforced, the carbohydrate chains were docked with the intermolecular distance restraints
NMR representativeSelection criteria: target function
NMR ensembleConformer selection criteria: structures with the least restraint violations
Conformers calculated total number: 200 / Conformers submitted total number: 16

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